The applicants' broad and long term objectives are to investigate excitation contraction (EC) coupling and in' particular mechanisms by which sarcoplasmic reticular (SR) Ca release is triggered in mammalian heart. Defects in the mechanism of EC coupling may be central to such health related problems as the long QT syndrome, heart failure and various cardiomypathies.
The specific aims mainly involve establishing that the relationship between trigger Ca and SR Ca release is non linear so that two separate triggers e.g. T type Ca current or Na-Ca exchange sum their effects with L type Ca current in a non linear fashion. Thus small triggers might have effects that are disproportionately large. Therefore the influence of small triggers could be significant in regulating SR Ca release.
The specific aims are 1) To investigate the relationships between SR Ca release triggers and SR Ca release 2) To investigate the relationship between Ca sparks and SR release triggers and 3) To investigate the effect of Na current on both macroscopic and microscopic gain.
These specific aims will be approached with the following basic research design and methods. The relationship between SR Ca release and Ca current will be measured at constant voltage using voltage clamp, fluorescent indicators (Fluo-3) and a rapid solution-changing device. The relationship between SR Ca release and Ca current will be established at several different but constant voltages. For example the relationship will be established at -10 mV and at +50 mV. From these relationships the macroscopic gain of the system will be calculated as the rate of SR Ca release divided by the magnitude of the L type Ca current. The effect of intracellular Na, Na current and reverse Na-Ca exchange on this relationship will be established. Ca sparks will be measured with a confocal microscope operating in line scan mode in both Rabbit and Mouse ventricular myocytes at 36 degrees C using the Ca indicator Fluo-3. The hypothesis that the probability of spark occurrence that is activated by Ca current can be influenced by Na-Ca exchange will be measured by comparing spark occurrence in transgenic mice overexpressing the Na-Ca exchange and wildtype mice which do not. The design of these experiments is such that sparks can be measured in a fixed location in one confocal plane thus greatly simplifying the analysis. Finally the effect of Na, Na-Ca exchange and controlled Na currents on microscopic gain will be tested.
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